* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download BBSRC 24/B11662 "Protein processing and electron transfer in
G protein–coupled receptor wikipedia , lookup
Gene expression wikipedia , lookup
NADH:ubiquinone oxidoreductase (H+-translocating) wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Interactome wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Catalytic triad wikipedia , lookup
Radical (chemistry) wikipedia , lookup
Point mutation wikipedia , lookup
Oxidative phosphorylation wikipedia , lookup
Homology modeling wikipedia , lookup
Biosynthesis wikipedia , lookup
Biochemistry wikipedia , lookup
Expression vector wikipedia , lookup
Protein purification wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Evolution of metal ions in biological systems wikipedia , lookup
Two-hybrid screening wikipedia , lookup
Western blot wikipedia , lookup
FULL REPORT Objectives 1&2 : to investigate the chemical mechanism of formation of the thioether bond at the active site through model compound and protein studies. to investigate the chemistry underlying pro-sequence processing events through mutagenesis and characterisation of resultant enzyme species. Galactose oxidase (GO) (EC 1.1.3.9) catalyses the oxidation of primary alcohols including D-galactose, to aldehydes. It is a monomeric, principally beta structure protein of three domains with a single copper atom and a protein-derived tyrosine radical as cofactors. It belongs to a growing class of proteins that self-catalyse assembly of their redox-active cofactors derived from active site amino acids. The enzyme exists in three redox states: reduced (Cu(I)-Tyr); semi-reduced (Cu(II)-Tyr) and oxidised (Cu(II)-Tyry) that are distinguishable by UV-vis spectroscopy. 41aa Precursor GO GO requires several post-translational modifications to generate the active enzyme; 1. cleavage of a secretion signal sequence, 2. cleavage of an N-terminal pro-sequence, 3. formation of a thioether bond between Cys228 and Tyr 272, and 4. oxidation to the radical state. An important tool in the analysis of processing is the resolution of distinct forms of GO by SDS-PAGE. This allows ProGO, Premat GO and mature GO to be distinguished by their distinct migration. Growth under copper limited conditions led to three species that were identified by N-terminal amino acid sequencing. Subsequently we perfected the process of preparing copper-free GO from Aspergillus. Processing step (1) will occur in the ER through signal peptidase action. To date, we have not studied this step. ss 18aa Post-ER GO PRO-GO (Aspergillus 639aa ? pro mature 6aa 17aa PRE-MAT GO (Pichia MAT GO Active MAT-GO Cys22 Tyr27 Y Tyr272 radical To investigate step (2) we have performed crystallographic analyses of proGO that reveal important interactions between the prosequence and residues in the mature portion of the protein. Regions of the protein involving residues (1 to 5, 189 to 200, 216 to 227, 254 to 261, 290 to 296) show a rmsd of 5.1Å compared with the mature enzyme structure. When the prosequence is removed, there are substantial changes in these regions of the protein with some residues that make interactions with the prosequence now interacting with other residues in the mature protein. There are major effects on the active site. For example Trp290 Cα moves by 6.3Å. Soaking experiments where copper was introduced into proGO crystals under anaerobic conditions reveal a bound copper with His496, His581 and Tyr495 as ligands, but the prosequence is retained and the thioether bond does not form. This suggests that processing can not occur in this crystal form under these conditions. We have recently generated an antibody that is specific for the prosequence by a peptide synthesis route. We have demonstrated that this antibody will cross react only with proGO and not with other forms of the protein lacking the prosequence. Use of this antibody to analyse samples of Aspergillus expressing GO reveal detection of GO both within the mycelium and in the supernatant. Notably the form of GO identified within the mycelium is the mature protein We see no proGO within the mycelium. This provides the first direct evidence for prosequence processing within the cell. It seems likely that the processing occurs quite rapidly following synthesis, accounting for undetectable steady-state levels of proGO. Speculatively we propose that processing is most likely to occur in the Golgi and experiments with secretion inhibitors, such as brefeldin A, are important to determine whether we can detect accumulation of proGO following imposition of secretion blocking agents. The anti-proGO antibody also provides a tool for the development of techniques to monitor prosequence cleavage in solution. To study steps (3) and (4), we have purified a premature form of GO (prematGO) from metal-free growth conditions, that does not contain the prosequence. We have previously used Aspergillus nidulans as the heterologous expression host for GO, which is naturally produced by the filamentous fungus Fusarium graminearum. The generation of transformants and the relative difficulty of handling Aspergillus and the time consuming nature of grow-ups led us to explore alternate expression systems for the processing studies. We therefore established an efficient Pichia pastoris system through subcloning the gene inframe with the mating factor secretion sequence in the vector pPICαZ (Invtirogen). This system relies upon induction of the aox promoter upon methanol addition to drive expression of the cloned transgene. We generated two version of the GO gene in this vector, one carrying only the mature sequence and used for the premature GO studies described below, and one carrying the mature sequence plus the 17 amino acid N-terminal prosequence (proGO). The proteins were produced and characterised by a range of techniques including ES-MS, N-terminal sequencing, ICP-MS for metal analysis, kinetics, UV-vis spectroscopy and crystallographic studies. The proteins behaved as anticipated with the exception of prosequence processing in the proGO. ES-MS and N-terminal sequencing indicated that prosequence processing did not occur in the same clean processing manner as in A. nidulans. The major species was the mature protein, but there is also a substantial contribution from a form with the residues -3 to -1 (SLR) of the prosequence. This finding has limited our ability to study prosequence processing in Pichia expressed enzyme; we have had to rely on the use of A. nidulans and so have not investigated the range of mutational variants we had intended to study. Most recently we have developed an expression system based on E. coli, in which a directed evolution form of GO with enhanced expression has been coupled with the inclusion of N-terminal synonymous mutations to provide very high level expression (~20 mg/L). This system produces the proGO intracellularly and therefore provides a good expression system for producing protein for studying processing steps. For our studies of step 4, we used the prematGO construct in Pichia under normal copper supplemented growth conditions and demonstrated that the 3d structure of the mature form is identical to that previously determined for other preparations of GO. We also performed spectroscopic and kinetic measurements that show the protein has essentially the same properties as protein produced in our original expression host Aspergillus nidulans. Defining appropriate metal-free purification conditions for the protein was not trivial and led to the understanding that protein quality can vary. By this we mean that premat GO can be purified that behaves normally in terms of thioether bond formation, but that will not oxidise when exposed to oxygen. This protein will however oxidise with other oxidants such as ferricyanide. This presumably reflects some subtle difference within the enzyme. It thus proved time-consuming to generate high quality copper-free enzyme for the various studies reported here. For the pro-form of GO, it is known that aerobic incubation in the presence of Cu(II) leads to pro-sequence cleavage, thioether bond formation and oxidation of the protein to the radical state. We have shown that prematGO can similarly process under aerobic conditions to give the active radical state consistent with it being a processing intermediate. In addition, under anaerobic conditions, the thioether bond forms efficiently but the enzyme is not oxidised to the radical state; the resulting protein can however be oxidised to the radical state by reaction with oxygen or Fe(CN)63-. Under anaerobic conditions addition of Cu(II) leads to the formation of a 410 nm species that may represent a processing intermediate. Exposure of this protein species to oxygen leads to loss of the 410nm feature and rapid production of the spectrum of oxidised GO (445 nm and broad 800 nm feature). We have also determined 3d structures under cryocrystallography conditions for the prematGO without Cu(II) and following brief (3min) soaking of apoform crystals with Cu(II) under anaerobic conditions. This reveals an unusual copper (II) co-ordination with Cys228, His496 and His581. This SN2 co-ordination of Cu(II) is supported by a range of other studies, including EXAFS, rR, EPR and comparision with a model Mop5 (Schnepf et al 2001 JACS 123, 2186) Longer incubation of the premat GO crystals with Cu(II) (24hr) reveals formation of the thioether bond. This supports the solution studies and provides further evidence that thioether bond formation can occur under anaerobic conditions. Recently Whittaker and Whittaker (2003, JBC 278, 22090–101) have reported processing of premat GO by Cu(I) and we have been able to reproduce these results. We have also shown that Cu(I) supports anaerobic formation of the thioether bond at a rate similar to Cu(II). These observations indicate that an alternate electron acceptor must exist for formation of the thioether bond since the exogenous electron acceptors (Cu(II) & oxygen) are absent. The bond is also able to form at stoichiometric and sub-stoichiometric concentrations of Cu(II) further implicating an alternate electron acceptor, most likely residues on the protein. One obvious potential site is the disulphide C515/C518 quite close to the active site. However, titration of cysteine residues within GO before and after processing provides no evidence for alteration of their redox state. At present we do not know the identity of the endogenous electron acceptor; however, in experiments performed under conditions of excess Cu(II), the rate of thioether bond formation increases with copper concentration suggesting that Cu(II) can act as an additional electron acceptor. We have recently solved the crystal structure of prematGO soaked with Cu(I) under anaerobic conditions and the thioether bond has formed within 30 min. Earlier time points will reveal important information about the initial Cu(I) co-ordination to prematGO and whether this differs from the SN2 coordination of Cu(II). None of the crystallographic evidence so far reveals copper co-ordination to Tyr272 making the involvement of a tyrosyl radical mechanism unlikely. We considered a sulphenic acid mechanism for bond formation since an oxidised Cys228 was seen during the structural studies of the proGO, but processing is still supported albeit more slowly, at pH9, while sulphenate mechanisms are not expected to operate above pH8.5. The observed interaction between Cys228 and Cu leads us to propose a thiyl radical mechanism for the formation of the thioether bond. B We have generated a number of mutational variants of D A C active site residues, in particular Y495A, H496A, H581A, Y272A to investigate the importance of copper binding at the active site on the processing of GO. One difficulty we have faced relates to the production of the proform of GO in Pichia pastoris (see above) The Y272A variant displays altered properties and Nterminal sequencing reveals the additional 6 amino acids that E precede the 17 amino acid region we call the prosequence. It is not yet clear why this mutation results in this failure to process in H G the presence of copper and further studies are necessary. F We have also investigated the role of the only free cysteine in GO, C383 that lies at the back of the active site. Mutation of this residue to serine leads to a 5-fold reduction in KM for GO, but the enzyme still form the thioether bond normally. Interestingly a C383A variant has a similar KM to wild type. We have determined the 3d structure of this mutational variant to investigate the cause of the reduction in KM but the structure does not provide a satisfactory explanation. Remarkably however, the structure was determined in the presence of –methyl-1,3-propandiol as cryoprotectant and this compound 410 nm Complex S Tyr 272 S- HS OH OH OH Cys 228 S OH H Cu(I) Cu(II) O2 Cu(II) OR HO2 X Cu(I)/H+ Seen in EPR O- H S Cu(II) e-/H+ S O Cu(II) S O OH Cu(I) S H Cu(I) is observed in the active site of the enzyme as a potential substrate. This is accompanied by the presence of an additional water in C383S compared to wild-type, that interacts with the OH of Tyr405 and therefore suggests an altered pattern of H-bonds that may be responsible for the enhanced substrate binding to this mutant enzyme. Objective 3 : to explore the nature and role of a putative electron transfer pathway between Cys515/Cys518 and the radical site in galactose oxidase by a combination of mutagenesis, biochemistry and spectroscopy. The studies on the processing aspects of the enzyme proved more time consuming than anticipated, and due to their exciting and topical nature, we focused more intently on these aspects than on the electron transfer aspect. Nonetheless, we have generated mutations at Cys515 and Cys518 to explore their effects on radical decay. Sykes and colleagues have performed pulse radiolysis experiments in which they claim to have identified a transient cysteinyl radical. They ascribed the site involved to be Cys515 or Cys518, but provided no direct evidence for this. They titrated the protein with thiophosphorylchloride and concluded that the modification (presumed to be at C515/518) stabilsed the radical species and prevented the normal rate of radical decay. This would be consistent with Cys515/Cys518 acting as an electron source during the decay process. We took a different, more direct approach and mutated these residues to Ala. The enzyme processes normally and retains enzyme activity, but the radical decays much more rapidly than in wild-type GO, and yet the disulphide bond is absent. In a single mutant C518S, the rapid also decay more rapidly than wild-type, but more slowly than in the double mutant. These data suggest that Cys515 and Cys518 are not involved in the radical decay pathway.. This conclusion contradicts that by Sykes and colleagues, although the experiments were admittedly different. Perhaps they were titrating a different cysteine, although this seems unlikely. Cys383 should not be accessible, and in any case we have shown by mutation, has no effect on radical stability. The second disulphide in GO is on domain 1 (Cys18/Cys27), a long way from the active site and therefore an unlikely source of electrons. Finally it is very unlikely that they would have modified Cys228 since this residue is involved in a thioether linkage. We can not exclude a role for C515/C518 in enzyme processing. Under physiological conditions there may be a wealth of exogenous oxidants capable of supporting thioether formation and radical oxidation. However, when we force the reaction to occur under non-physiological in vitro anaerobic conditions, then perhaps the process occurs by another pathway, perhaps involving these cysteines or other residues within the protein. A new galactose oxidase We have cloned and are beginning to characterise the GO homologue from F. veneatum that shows distinct differences in substrate specificity and spectral properties. We are going to investigate the presence of a thioether bond and whether this enzyme also undergoes similar post-translational cofactor processing. Issues relating to the grant The award of a single PDRA precluded any work on model compounds and therefore the work focused on protein studies. The project started in Nov 1999 with the appointment of Dr C Allardyce. She worked on the grant for 8 months before leaving to move to London for personal reasons. The grant was put into abeyance for 6 months pending the appointment of a new PDRA. We believe that we have met many of the objectives set out in the original application and given the constraints of reduced funding level and staff retention and reappointment issues, and the technical difficulties associated with production of high quality copper-free precursor protein. These have led to some delay in publication of our work that does not reflect the achievements made during this grant. The work advances our understanding of the posttranslational modification of an amino acid derived cofactor involved in the expansion of biological catalytic capability. Such understanding is important for future manipulation of biocatalysts and should allow us to tailor enzymes to perform new chemistries that are currently difficult with enzyme catalysts. The general area is one in which there is considerable international interest and we regularly participate in the Gordon Conference dedicated to this scientific area. The project has extended our collaboration with Prof. D M Dooley in Bozeman, Montana and we have also has some collaboration with scientists at Hercules Inc. (Delaware and Barnveld, The Netherlands) who are interested in industrial applications of GO as a biocatalyst. We have recently prepared manuscripts describing both solution and structural studies of processing in prematGO and the characterisation of the C383S variant. • D. Wilkinson, N. Akumanyi, R. Hurtado Guerrero, H. Dawkes, P. F. Knowles, S. E. V. Phillips and M. J.McPherson Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution, Submitted to Protein Engineering. • R. Hurtado-Guerrero, M.S. Rogers, S. J. Firbank, D.M. Dooley<, M.A. Halcrow, S. E. V. Phillips, P.F. Knowles and M.J. McPherson Formation of the thioether bond in galactose oxidase does not require oxygen. To be submitted to Journal of Biological Chemistry The work has been presented at several international meetings. • Galactose oxidase post-translational processing. 54th Harden Conference on “Enzymology: emerging trends and future prospects”, Ambleside, UK, 2002. y Cofactor processing and catalysis in galactose oxidase, CERLIB, Chamonix, France, 2003 y Cofactor processing and catalysis in galactose oxidase, Enzymology: a structural perspective, Biochemical Society Focus Meeting, St. Andrews University, 2003 y Cofactor processing in galactose oxidase, Biochemical Society Annual Symposium, Essex, 2003.